Thrombosis is the formation, development, or existence of a blood clot or thrombus within the vascular system. This is a life saving event when occurs during hemorrhage and a life threatening event when it occurs at any other time. Occlusion of a blood vessel, caused by excessive platelet activation (by the stimulation of an agonist) and recruitment (leading to platelet aggregation and vessel occlusion), are the major contributing factors in clinical disorders such as stroke, myocardial infarction, unstable angina, and restenosis. Therefore, there is a great need to identify therapeutic strategies and compositions for the pharmacological neutralization of platelet reactivity (activation, recruitment, and aggregation).
Currently, several treatment strategies are available to deal with a thrombus formation and fall into two classes, pharmacological (protein based and small molecule bases) and mechanical (for example, percutaneous coronary intervention (PCI), bypass surgery, sonication). The pharmacological classes of treatments cover several therapeutic approaches such as: acting as anti-coagulants (for example, heparin and hirudin), thrombolytic agents (for example, tPA, pro-urokinase, and Streptokinase) or antiplatelet agents (for example, aspirin, ticlopidine, and clopidogel). However, their therapeutic utility is limited due to a significant risk of bleeding complications (Wardlaw, J. M., et al., Lancet (1997) 350:607-614). For example, in the United States, less than 2% of the patients with acute ischemic stroke can receive rtPA due to high intracranial hemorrhage risks (Chiu, D., et al., Stroke (1998) 29:18-22). Glycoprotein IIb/IIIa antagonists such as ReoPro® (monoclonal antibody) have been used for PCI and are currently under clinical investigation for the treatment of patients with acute coronary syndromes and acute ischemic stroke. However, the inhibition of the glycoprotein IIb/IIIa receptors will interfere with platelet adhesion and clot retraction resulting in bleeding complications. Therefore, it is important to identify novel strategies for inhibition of platelet activation and aggregation that will cause minimal risks of bleeding.
During early stages of platelet activation, several agonist including ADP, Thromboxane A2 and serotonin are released. Among these, ADP is the single most important platelet agonist and recruiting agent that is present in the thrombus microenvironment (Marcus, A. J. and Safier, L. B., FASEB J. (1993) 7:516-522). Part of the normal function of endothelial cells ability to maintain blood fluidity is the local generation of an enzyme with ectoapyrase (apyrase, ATP diphosphohydrolase, ATP-diphosphatase, Adenosine diphosphatase, ADPase, E-NTPDase, EC 3.6.1.5) activity such as CD39. CD39 is a constitutively expressed enzyme having apyrase activity that strongly inhibits platelet aggregation by rapidly metabolizing ADP released from activated platelets, thus terminating further platelet recruitment and aggregation. (Marcus, A. J., et al., J. Clin. Invest. (1997) 99:1351-1360; Gayle, R., et al., J. Clin. Invest (1998) 101:1851-1859). Several research studies have now established CD39 as the prime thromboregulator (Marcus, A. J., et al., supra; Kaczmarek, E., et al., J. Biol. Chem. (1996) 271:33116-33122). In addition, animal model studies indicate that administration of a soluble form of CD39 for treatment has significant clinical advantages over existing treatment regimes without the life threatening bleeding complications often associated with the current treatment strategies (PCT WO 01/11949; PCT WO 00/23459).
This invention is directed to the use of ADPase enhanced apyrases, useful for the inhibition of platelet activation and as a general thromboregulator useful for the treatment and prevention of stroke and other diseases involving thrombosis.